PROJECT SUMMARY/ABSTRACT Preservation of mobility is one of the most significant health concerns of the elderly population associated with aging. Identification of cost-effective therapies that can preserve independence and mobile capacity in the elderly would be important and influence many other aspects of mental and physical health. Even though endurance exercise is the most effective intervention that can reproducibly extend mobile healthspan in humans and animals, the molecular mechanism underlying exercise benefits has been elusive. I propose to investigate the role of a potential exercise mediator Sestrins, which may lead to the development of therapeutic or preventive methods that can harness the benefits of exercise without actual physical movement, which is practically challenging in this modern society. Sestrins are a family of stress-inducible proteins that can suppress both reactive oxygen species (ROS) and mTOR complex 1 (mTORC1) signaling pathways. Excessive ROS accumulation and chronic mTORC1 activation are known as promoters of tissue aging and degeneration. Genetic studies in worms, flies, and mice demonstrated that loss of Sestrins accelerates various age- and obesity-associated metabolic pathologies including cardiac dysfunction, insulin resistance and muscle degeneration, associated with ROS accumulation and hyperactive mTORC1. Interestingly, while Sestrins downregulate ROS and mTORC1, they induce autophagy and strongly potentiate mTORC2/AKT signaling activation in response to insulin. The potential anti-aging properties of Sestrins prompted me to investigate whether we are able to attenuate tissue aging by transgenic supplementation of Sestrins activity. Preliminary experiments in Drosophila indicated that Sestrin overexpression in muscle tissue can dramatically increase mobility in aged flies: a more than twofold increase in both climbing speed and endurance was observed in Sestrin-overexpressing flies compared to the control group. Exercise training in both flies and mice can increase Sestrin expression. Loss of Sestrin in flies and Sestrin1 in mice nullified the benefits received from exercise training in expanding their endurance and improving their metabolism. Based on these findings, we propose to (aim 1) characterize skeletal, cardiac and diaphragm muscle phenotypes of Sestrins-deficient mice across ages, (aim 2) characterize muscle phenotypes of mice with muscle-specific Sestrins modulation, and (aim 3) examine the mechanism of how Sestrins improve muscle functionality. Our central hypothesis is that, as in flies, mammalian Sestrins are also important exercise mediators and can produce mobility-extending activities in later life. Therefore, the proposed research may reveal the beneficial effects of Sestrin activation in protection against aging and age-associated functional and structural degeneration of skeletal and cardiac muscle. This may lead to the development of innovative Sestrins-mimicking methods for preserving mobility and improving quality of life in later ages.